Osmotically stabilized microbubble preparations

ABSTRACT

A microbubble preparation formed of a plurality of microbubbles comprising a first gas and a second gas surrounded by a membrane such as a surfactant, wherein the first gas and the second gas are present in a molar ratio of from about 1:100 to about 1000:1, and wherein the first gas has a vapor pressure of at least about (760-x) mm Hg at 37° C., where x is the vapor pressure of the second gas at 37° C., and wherein the vapor pressure of each of the first and second gases is greater than about 75 mm Hg at 37° C.; also disclosed are methods for preparing microbubble compositions, including compositions that rapidly shrink from a first average diameter to a second average diameter less than about 75% of the first average diameter and are stabilized at the second average diameter; kits for preparing microbubbles; and methods for using such microbubbles as ultrasound contrast agents.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/863,982 filed May 21, 2001, now U.S. Pat. No. 6,706,253, which is acontinuation of U.S. patent application Ser. No. 08/785,007 filed Jan.17, 1997, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 08/405,447, filed Mar. 16, 1995, now U.S. Pat. No.5,639,443, which is a continuation of U.S. patent application Ser. No.08/099,951, filed Jul. 30, 1993 now abandoned.

FIELD OF THE INVENTION

The present invention includes a method for preparing stable long-livedmicrobubbles for ultrasound contrast enhancement and other uses, and tocompositions of the bubbles so prepared.

BACKGROUND OF THE ART

Ultrasound technology provides an important and more economicalalternative to imaging techniques which use ionizing radiation. Whilenumerous conventional imaging technologies are available, e.g., magneticresonance imaging (MRI), computerized tomography (CT), and positronemission tomography (PET), each of these techniques use extremelyexpensive equipment. Moreover, CT and PET utilize ionizing radiation.Unlike these techniques, ultrasound imaging equipment is relativelyinexpensive. Moreover, ultrasound imaging does not use ionizingradiation.

Ultrasound imaging makes use of differences in tissue density andcomposition that affect the reflection of sound waves by those tissues.Images are especially sharp where there are distinct variations intissue density or compressibility, such as at tissue interfaces.Interfaces between solid tissues, the skeletal system, and variousorgans and/or tumors are readily imaged with ultrasound.

Accordingly, in many imaging applications ultrasound performs suitablywithout use of contrast enhancement agents; however, for otherapplications, such as visualization of flowing blood in tissues, therehave been ongoing efforts to develop such agents to provide contrastenhancement. One particularly significant application for such contrastagents is in the area of vascular imaging. Such ultrasound contrastagents could improve imaging of flowing blood in the heart, kidneys,lungs, and other tissues. This, in turn, would facilitate research,diagnosis, surgery, and therapy related to the imaged tissues. A bloodpool contrast agent would also allow imaging on the basis of bloodcontent (e.g., tumors and inflamed tissues) and would aid in thevisualization of the placenta and fetus by enhancing only the maternalcirculation.

A variety of ultrasound contrast enhancement agents have been proposed.The most successful agents have generally consisted of microbubbles thatcan be injected intravenously. In their simplest embodiment,microbubbles are miniature bubbles containing a gas, such as air, andare formed through the use of foaming agents, surfactants, orencapsulating agents. The microbubbles then provide a physical object inthe flowing blood that is of a different density and a much highercompressibility than the surrounding fluid tissue and blood. As aresult, these microbubbles can easily be imaged with ultrasound.

Most microbubble compositions have failed, however, to provide contrastenhancement that lasts even a few seconds, let alone minutes, ofcontrast enhancement. This greatly limits their usefulness. Microbubbleshave therefore been “constructed” in various manners in an attempt toincrease their effective contrast enhancement life. Various avenues havebeen pursued: use of different surfactants or foaming agents; use ofgelatins or albumin microspheres that are initially formed in liquidsuspension, and which entrap gas during solidification; and liposomeformation. Each of these attempts, in theory, should act to createstronger bubble structures. However, the entrapped gases (typically air,CO₂, and the like) are under increased pressure in the bubble due to thesurface tension of the surrounding surfactant, as described by theLaPlace equation (ΔP=2γ/r).

This increased pressure, in turn, results in rapid shrinkage anddisappearance of the bubble as the gas moves from a high pressure area(in the bubble) to a lower pressure environment (in either thesurrounding liquid which is not saturated with gas at this elevatedpressure, or into a larger diameter, lower pressure bubble).

Solid phase shells that encapsulate gases have generally proven toofragile or to permeable to the gas to have satisfactory in vivo life.Furthermore, thick shells (e.g., albumin, sugar, or other viscousmaterials) reduce the compressibility of the bubbles, thereby reducingtheir echogenicity during the short time they can exist. Solid particlesor liquid emulsion droplets that evolve gas or boil when injected posethe danger of supersaturating the blood with the gas or vapor. This willlead to a small number of large embolizing bubbles forming at the fewavailable nucleation sites rather than the intended large number ofsmall bubbles.

One proposal for dealing with such problems is outlined in Quay,PCT/US92/07250. Quay forms bubbles using gases selected on the basis ofbeing a gas at body temperature (below 37° C.), and having reduced watersolubility, higher density, and reduced gas diffusivity in solution incomparison to air. Although reduced water solubility and diffusivity canaffect the rate at which the gas leaves the bubble, numerous problemsremain with the Quay bubbles. Forming bubbles of sufficiently smalldiameter (e.g., 0.2 μm) requires high energy input. This is adisadvantage in that sophisticated bubble preparation systems must beprovided at the site of use. Moreover, The Quay gas selection criteriaare incorrect in that they fail to consider certain major causes ofbubble shrinkage, namely, the effects of bubble surface tension,surfactants and gas osmotic effects, and these errors result in theinclusion of certain unsuitable gases and the exclusion of certainoptimally suitable gases.

Accordingly, a need exists in the art for compositions, and a method toprepare such compositions, that provide, or utilize, a longer lifecontrast enhancement agent that is biocompatible, easily prepared, andprovides superior contrast enhancement in ultrasound imaging.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided anultrasound contrast enhancement agent that has a prolonged longevity invivo, which consists of virtually any conventional microbubbleformulation in conjunction with an entrapped gas or gas mixture that isselected based upon consideration of partial pressures of gases bothinside and outside of the bubble, and on the resulting differences ingas osmotic pressure that oppose bubble shrinkage. Gases having a lowvapor pressure and limited solubility in blood or serum (i.e.,relatively hydrophobic) may advantageously be provided in combinationwith another gas that is more rapidly exchanged with gases present innormal blood or serum. Surfactant families allowing the use of highermolecular weight gas osmotic agents, and improved methods of bubbleproduction are also disclosed.

One aspect of the present invention is a stabilized gas filledmicrobubble preparation, comprising a mixture of a first gas or gasesand a second gas or gases within generally spherical membranes to formmicrobubbles, wherein the first gas and the second gas are respectivelypresent in a molar ratio of about 1:100 to about 1000:1, and wherein thefirst gas has a vapor pressure of at least about (760-x) mm Hg at 37°C., where x is the vapor pressure of the second gas at 37° C., andwherein the vapor pressure of each of the first and second gases isgreater than about 75 mm Hg at 37° C., with the proviso that the firstgas and the second gas are not water vapor. In one embodiment, thesecond gas comprises a fluorocarbon and the first gas is anonfluorocarbon, such as nitrogen, oxygen, carbon dioxide, or a mixturethereof.

The microbubbles may advantageously be provided in a liquid medium, suchas an aqueous medium, wherein they have a first average diameter, theratio of the first gas to the second gas in the microbubbles is at least1:1, and the microbubbles are adapted to shrink in the medium as aresult of loss of the first gas through the membrane to a second averagediameter of less than about 75% of the first diameter and then remainstabilized at or about the second diameter for at least about 1 minuteas a result of a gas osmotic pressure differential across the membrane.Advantageously, the medium is in a container and the microbubbles haveactually been formed in the container. Alternatively, the medium isblood in vivo. In one embodiment, the liquid medium contains gas orgases dissolved therein with a gas tension of at least about 700 mm Hg,wherein the first diameter is at least about 5 μm, and wherein thetension of the gas or gases dissolved in the medium is less than thepartial pressure of the same gas or gases inside the microbubbles.

In a particularly preferred embodiment, the bubble initially contains atleast three gases: a first gas having a partial pressure far greaterthan the gas tension of the same gas in the surrounding liquid (e.g.,1.5, 2, 4, 5, 10, 20, 50, or 100 or more times greater than in thesurrounding liquid); a second gas that is retained in the bubble due toa relatively low permeability of the bubble membrane to the gas, or arelatively low solubility of the gas in the surrounding medium (asdescribed elsewhere herein), and a third gas to which the membrane isrelatively permeable that is also present in the surrounding medium. Forexample, in an aqueous system exposed to or at least partiallyequilibrated with air (such as blood), the first gas may advantageouslybe carbon dioxide or another gas not present in large quantities in airor blood; the second gas may be a fluorocarbon gas, such asperfluorohexane; and the third gas may be air or a major component ofair such as nitrogen or oxygen.

Preferably, the first diameter prior to shrinkage is at least about 10μm and the second diameter at which the diameter is stabilized isbetween about 1 μm and 6 μm.

For all of the microbubble preparations or methods described herein, inone preferred embodiment, the second gas has an average molecular weightat least about 4 times that of the first gas. In another preferredembodiment, the second gas has a vapor pressure less than about 750 or760 mm Hg at 37° C. Moreover, it is preferred that the molar ratio ofthe first gas to the second gas is from about 1:10 to about 500:1,200:1, or 100:1. In other preferred embodiments, the second gascomprises a fluorocarbon or a mixture of at least two or threefluorocarbons, and the first gas is a nonfluorocarbon. In someadvantageous preparations, the second gas comprises one or moreperfluorocarbons. In others, both the first gas and the second gascomprise fluorocarbons. In still others, the microbubbles contain as thefirst gas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluorohexane in a ratio from about1:10 to about 10:1. Alternatively, the microbubbles contain as the firstgas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluoropentane in a ratio fromabout 1:10 to about 10:1. It is advantageous that the second gas leavethe microbubble much more slowly than does the first gas; thus, it ispreferred that the second gas has a water solubility of not more thanabout 0.5 mM at 25° C. and one atmosphere, and the first gas has a watersolubility at least about 10 times, and preferably at least 20, 50, 100,or 200 times greater than that of the second gas. Similarly, it ispreferred that the permeability of the membrane to the first gas is atleast about 5 times, preferably 10, 20, 50, or 100 times greater thanthe permeability of the membrane to the second gas.

The microbubble preparation may advantageously be contained in acontainer, having a liquid in the container in admixture with themicrobubbles, wherein the container further comprises means fortransmission of sufficient ultrasonic energy to the liquid to permitformation of the microbubbles by sonication. In this way, themicrobubbles can be formed by the physician (or other professional)immediately before use by applying ultrasonic energy from an outsidesource to the sterile preparation inside the container. This means fortransmission can, for example, be a flexible polymer material having athickness less than about 0.5 mm (which permits ready transmission ofultrasonic energy without overheating the membrane). Such membranes canbe prepared from such polymers as natural or synthetic rubber or otherelastomer, polytetrafluoroethylene, polyethylene terephthalate, and thelike.

In the microbubble preparations of the invention, the membrane enclosingthe gas is preferably a surfactant. One preferred type of surfactantcomprises a non-Newtonian viscoelastic surfactant, alone or incombination with another surfactant. Other preferred general andspecific categories of surfactants include carbohydrates, such aspolysaccharides, derivatized carbohydrates, such as fatty acid esters ofsugars such as sucrose (preferably sucrose stearate), and proteinaceoussurfactants including albumin. Alternatively, the membrane of themicrobubble need not be a fluid (such as a surfactant), but instead canbe a solid or semi-solid, such as hardened, thickened, or denaturedproteinaceous material (e.g. albumin), carbohydrates, and the like.

One advantageous form of the invention is a kit for use in preparingmicrobubbles, preferably at the site of use. This kit may comprisesealed container (such as a vial with a septum seal for easy removal ofthe microbubbles using a hypodermic syringe), a liquid in the container(such as water or a buffered, isotonic, sterile aqueous medium), asurfactant in the container, and a fluorocarbon gas (including afluorocarbon vapor) in the container, wherein the liquid, thesurfactant, and the fluorocarbon gas or vapor are together adapted toform microbubbles upon the application of energy thereto. The energyadvantageously may be simple shaking energy, either manual ormechanical, stirring or whipping, or ultrasonic energy. The kitpreferably includes means in the container for permitting transmissionof sufficient external ultrasonic energy to the liquid to formmicrobubbles in the container. As above, the means for transmission canin one embodiment comprise a flexible polymer membrane having athickness less than about 0.5 mm. In one embodiment, the kit furtherincludes a nonfluorocarbon gas in the container, wherein the molar ratioof the nonfluorocarbon gas to the fluorocarbon gas is from about 1:10 toabout 1000:1, with the proviso that the nonfluorocarbon gas is not watervapor. In all of the kits of the present invention, the surfactant, thegas or gases, and the other elements of the kit may in some embodimentsbe the same as recited above for the microbubble preparation per se.

In another embodiment, the kit comprises a container, driedliquid-soluble void-containing structures in the container, thevoid-containing structures defining a plurality of voids having anaverage diameter less than about 100 μm, a gas in the voids, and asurfactant, wherein the void-containing structures, the gas, and thesurfactant are together adapted to form microbubbles upon addition tothe container of a liquid in which the void-containing structures aresoluble. These void-containing structures can be made at least in partof the surfactant, e.g., by lyophilization of void-forming material orby spray drying, or can be formed from any other liquid soluble(preferably water soluble) film-forming material, such as albumin,enzymes, or other proteins, simple or complex carbohydrates orpolysaccharides, and the like. The surfactants used in the kit canadvantageously be those described above in connection with themicrobubble preparations per se.

The present invention also includes a method for forming microbubbles,comprising the steps of providing a first gas, a second gas, a membraneforming material, and a liquid, wherein the first gas and the second gasare present in a molar ratio of from about 1:100 to about 1,000:1, andwherein the first gas has a vapor pressure of at least about (760-x) mmHg at 37° C., where x is the vapor pressure of the second gas at 37° C.,and wherein the vapor pressure of each of the first and second gases isgreater than about 75 mm Hg at 37° C., with the proviso that the firstgas and the second gas are not water vapor, and surrounding the firstand second gases with the membrane forming material to form microbubblesin the liquid. The membrane forming materials and gases may be asdescribed above. The method preferably further comprises the steps ofinitially forming microbubbles having a first average diameter whereinthe initial ratio of the first gas to the second gas in the microbubblesis at least about 1:1, contacting the microbubbles having a firstaverage diameter with a liquid medium, shrinking the microbubbles in themedium as a result of loss of the first gas through the membrane, andthen stabilizing the microbubbles at a second average diameter of lessthan about 75% of the first diameter for a period of at least oneminute. Preferably, the microbubbles are stabilized at the seconddiameter by providing a gas osmotic pressure differential across themembrane such that the tension of a gas or gases dissolved in the mediumis greater than or equal to the pressure of the same gas or gases insidethe microbubbles. In one embodiment, the first diameter is at leastabout 5 μm.

The invention also includes a method for forming microbubbles,comprising the steps of providing dried liquid-soluble void-containingstructures, the void-containing structures defining a plurality of voidshaving a diameter less than about 100 μm, providing a gas in the voids,providing a surfactant, combining together the void-containingstructures, the gas, the surfactant, and a liquid in which thevoid-containing structures are soluble, and dissolving thevoid-containing structures in the liquid whereby the gas in theenclosures forms microbubbles that are surrounded by the surfactant. Aswith the kit, preferred void-containing structures are formed ofprotein, surfactant, carbohydrate, or any of the other materialsdescribed above.

Finally, the present invention includes a method for imaging an objector body, comprising the steps of introducing into the object or body anyof the aforementioned microbubble preparations and then ultrasonicallyimaging at least a portion of the object or body. Preferably, the bodyis a vertebrate and the preparation is introduced into the vasculatureof the vertebrate. The method may further include preparing themicrobubbles in any of the aforementioned manners prior to introductioninto the animal.

DETAILED DESCRIPTION OF THE INVENTION

As used in the present description and claims, the terms “vapor” and“gas” are used interchangeably. Similarly, when referring to the tensionof dissolved gas in a liquid, the more familiar term “pressure” may beused interchangeably with “tension.” “Gas osmotic pressure” is morefully defined below, but in a simple approximation can be thought of asthe difference between the partial pressure of a gas inside amicrobubble and the pressure or tension of that gas (either in a gasphase or dissolved in a liquid phase) outside of the microbubble, whenthe microbubble membrane is permeable to the gas. More precisely, itrelates to differences in gas diffusion rates across a membrane. Theterm “membrane” is used to refer to the material surrounding or defininga microbubble, whether it be a surfactant, another film forming liquid,or a film forming solid or semisolid. “Microbubbles” are considered tobe bubbles having a diameter between about 0.5 and 300 μm, preferablyhaving a diameter no more than about 200, 100, or 50 μm, and forintravascular use, preferably not more than about 10, 8, 7, 6, or 5 μm(measured as average number weighted diameter of the microbubblecomposition). When referring to a “gas,” it will be understood thatmixtures of gases together having the requisite property fall within thedefinition, except where the context otherwise requires. Thus, air maytypically be considered a “gas” herein.

The present invention provides microbubbles that have a prolongedlongevity in vivo that are suitable for use as ultrasound contrastenhancement agents. Typical ultrasound contrast enhancement agentsexhibit contrast enhancement potential for only about one pass throughthe arterial system, or a few seconds to about a minute, and thus do notsurvive past the aorta in a patient following intravenous injection. Incomparison, contrast agents prepared in accordance with the presentinvention continue to demonstrate contrast enhancements lives measuredin multiple passes through the entire circulatory system of a patientfollowing intravenous injection. Bubble lives of several minutes areeasily demonstrated. Such lengthening of contrast enhancement potentialduring ultrasound is highly advantageous. In addition, the contrastenhancement agents of the invention provide superior imaging; forexample, clear, vivid, and distinct images of blood flowing through theheart, lungs, and kidneys are achieved. Thus small, nontoxic doses canbe administered in a peripheral vein and used to enhance images of theentire body.

While bubbles have been shown to be the most efficient ultrasoundscatterers for use in intravenous ultrasound contrast agents, their mainpractical drawback is the extremely short lifetime of the small(typically less than 5 microns diameter) bubbles required to passthrough capillaries in suspension. This short lifetime is caused by theincreased gas pressure inside the bubble, which results from the surfacetension forces acting on the bubble. This elevated internal pressureincreases as the diameter of the bubble is reduced. The increasedinternal gas pressure forces the gas inside the bubble to dissolve,resulting in bubble collapse as the gas is forced into solution. TheLaPlace equation, ΔP=2γ/r, (where ΔP is the increased gas pressureinside the bubble, γ is the surface tension of the bubble film, and r isthe radius of the bubble) describes the pressure exerted on a gas bubbleby the surrounding bubble surface or film. The LaPlace pressure isinversely proportional to the bubble radius; thus, as the bubbleshrinks, the LaPlace pressure increases, increasing the rate ofdiffusion of gas out of the bubble and the rate of bubble shrinkage.

It was surprisingly discovered that gases and gas vapor mixtures whichcan exert a gas osmotic pressure opposing the LaPlace pressure cangreatly retard the collapse of these small diameter bubbles. In general,the invention uses a primary modifier gas or mixture of gases thatdilute a gas osmotic agent to a partial pressure less than the gasosmotic agent's vapor pressure until the modifier gas will exchange withgases normally present in the external medium. The gas osmotic agent oragents are generally relatively hydrophobic and relatively bubblemembrane impermeable and also further possess the ability to develop gasosmotic pressures greater than 75 or 100 Torr at a relatively low vaporpressure.

The process of the invention is related to the well known osmotic effectobserved in a dialysis bag containing a solute that is substantiallymembrane impermeable (e.g. PEG, albumin, polysaccharide, starch)dissolved in an aqueous solution is exposed to a pure water externalphase. The solute inside the bag dilutes the water inside the bag andthus reduces the rate of water diffusion out of the bag relative to therate of pure water (full concentration) diffusion into the bag. The bagwill expand in volume until an equilibrium is established with anelevated internal pressure within the bag which increases the outwarddiffusional flux rate of water to balance the inward flux rate of thepure water. This pressure difference is the osmotic pressure between thesolutions.

In the above system, the internal pressure will slowly drop as thesolute slowly diffuses out of the bag, thus reducing the internal soluteconcentration. Other materials dissolved in the solution surrounding thebag will reduce this pressure further, and, if they are more effectiveor at a higher concentration, will shrink the bag.

It was observed that bubbles of air saturated with selectedperfluorocarbons grow rather than shrink when exposed to air dissolvedin a liquid due to the gas osmotic pressure exerted by theperfluorocarbon vapor. The perfluorocarbon vapor is relativelyimpermeable to the bubble film and thus remains inside the bubble. Theair inside the bubble is diluted by the perfluorocarbon, which acts toslow the air diffusion flux out of the bubble. This gas osmotic pressureis proportional to the concentration gradient of the perfluorocarbonvapor across the bubble film, the concentration of air surrounding thebubble, and the ratio of the bubble film permeability to air and toperfluorocarbon.

As discussed above, the LaPlace pressure is inversely proportional tothe bubble radius; thus, as the bubble shrinks, the LaPlace pressureincreases, increasing the rate of diffusion of gas out of the bubble andthe rate of bubble shrinkage, and in some cases leading to thecondensation and virtual disappearance of a gas in the bubble as thecombined LaPlace and external pressures concentrate the osmotic agentuntil its partial pressure reaches the vapor pressure of liquid osmoticagent.

We have discovered that conventional microbubbles that contain anysingle gas will subsist in the blood for a length of time that dependsprimarily on the arterial pressure, the bubble diameter, the membranepermeability of the gas through the bubble's surface, the mechanicalstrength of the bubble's surface, the presence, absence, andconcentration of the gases that are ordinarily present in the blood orserum, and the surface tension present at the surface of the bubble(which is primarily dependent on the diameter of the bubble andsecondarily dependent on the identity and concentration of thesurfactants which form the bubble's surface). Each of these parametersare interrelated, and they interact in the bubble to determine thelength of time that the bubble will last in the blood.

The present invention includes the discovery that a single gas or acombination of gases can together act to stabilize the structure of themicrobubbles entraining or entrapping them. Essentially, the inventionutilizes a first gas or gases (a “primary modifier gas”) that optionallyis ordinarily present in normal blood and serum in combination with oneor more additional second gases (a “gas osmotic agent or agents” or a“secondary gas”) that act to regulate the osmotic pressure within thebubble. Through regulating the osmotic pressure of the bubble, the gasosmotic agent (defined herein as a single or mixture of chemicalentities) exerts pressure within the bubble, aiding in preventingdeflation. Optionally, the modifier gas may be a gas that is notordinarily present in blood or serum. However, the modifier gas must becapable of diluting and maintaining the gas osmotic agent or agents at apartial pressure below the vapor pressure of the gas osmotic agent oragents while the gases in blood or other surrounding liquid diffuse intothe bubble. In an aqueous medium, water vapor is not considered to beone of the “gases” in question. Similarly, when microbubbles are in anonaqueous liquid medium, the vapor of that medium is not considered tobe one of the “gases.”

We have discovered that by adding a gas osmotic agent that has, forexample, a reduced membrane permeability through the bubble's surface orreduced solubility in the external continuous phase liquid phase, thelife of a bubble formed therewith will be radically increased. Thisstabilizing influence can be understood more readily through adiscussion of certain theoretical bubbles. First, we will consider theeffects of arterial pressure and surface tension on a hypotheticalmicrobubble containing only air.

Initially, a hypothetical bubble containing only air is prepared. Forpurposes of discussion, this bubble will initially be considered to haveno LaPlace pressure. Generally, when equilibrated at standardtemperature and pressure (STP), it will have a internal pressure of 760Torr of air and the surrounding fluid air concentration will also beequilibrated at 760 Torr (i.e., the fluid has an air tension of 760Torr). Such a bubble will neither shrink nor grow.

Next, when the above hypothetical bubble is introduced into the arterialsystem, the partial pressure of air (or air tension) in the blood (theair pressure at which the blood was saturated with air) will also beapproximately 760 Torr and there will be an arterial pressure (for thepurposes of the this discussion at 100 Torr). This total creates anexternal pressure on the bubble of 860 Torr, and causing the gases inthe bubble to be compressed until the internal pressure increases to 860Torr. There then arises a difference of 100 Torr between the airpressure inside the bubble and the air tension of the fluid surroundingthe bubble. This pressure differential causes air to diffuse out of thebubble, through its air-permeable surface membrane, causing the bubbleto shrink (i.e., lose air) as it strives to reach equilibrium. Thebubble shrinks until it disappears.

Next, consider the additional, and more realistic, effect on thehypothetical bubble of adding the surface tension of the bubble. Thesurface tension of the bubble leads to a LaPlace pressure exerted on gasinside the bubble. The total pressure exerted on the gas inside thebubble is computed through adding the sum of the atmospheric pressure,the arterial pressure and the LaPlace pressure. In a 3 μm bubble asurface tension of 10 dynes per centimeter is attainable with wellchosen surfactants. Thus, the LaPlace pressure exerted on thehypothetical 3 μm bubble is approximately 100 Torr and, in addition, thearterial pressure of 100 Torr is also exerted on the bubble. Therefore,in our hypothetical bubble, the total external pressure applied to thegas inside the bubble is 960 Torr.

The bubble will be compressed until the pressure of the air inside thebubble rises to 960 Torr. Accordingly, a concentration differential of200 Torr arises between the air inside the bubble and the air dissolvedin the blood. Therefore, the bubble will rapidly shrink and disappeareven more rapidly than it did in the previous case, as it attempts toreach equilibrium.

The discovery of the present invention is illustrated by considering athird hypothetical microbubble containing air and a gas osmotic agent ora secondary gas. Assume that a theoretical bubble, initially having noarterial pressure and no LaPlace pressure, is prepared having a totalpressure of 760 Torr, which is made up of air at a partial pressure of684 Torr and a perfluorocarbon (“PFC”) as a gas osmotic agent at apartial pressure of 76 Torr. Further, assume that the perfluorocarbon isselected to have one or more traits that make it capable of acting as anappropriate gas osmotic agent, such as limited bubble membranepermeability or limited solubility in the external liquid phase. Thereis an initial gas osmotic pressure differential between the 684 Torr ofair within the bubble and the 760 Torr of air tension outside the bubble(assuming STP) of 76 Torr. This 76 Torr initial pressure difference isthe initial gas osmotic pressure and will cause the bubble to expand.Air from outside of the bubble will diffuse into and inflate the bubble,driven by the osmotic pressure differential, similar to the way waterdiffuses into a dialysis bag containing a starch solution, and inflatesthe bag.

The maximum gas osmotic pressure this gas mixture can develop is relatedto the partial pressure of the PFC and the ratio of the permeability ofthe PFC to the permeability of the air in the surrounding fluid. Intheory, and as observed experimentally, the bubble will growindefinitely as the system attempts to reach osmotic equilibrium betweenthe concentration of air (equivalent to the partial pressure of air)within the bubble and the concentration of air surrounding the bubble(the air tension).

When the hypothetical mixed gas bubble is exposed to 100 Torr ofarterial pressure where the blood has a dissolved air tension of 760Torr, the total external pressure will equal 860 Torr (760 Torratmospheric pressure and 100 Torr arterial pressure). The bubble willcompress under the arterial pressure, causing the internal pressure ofthe bubble to reach 860 Torr. The partial pressure of the air willincrease to 774 Torr and the partial pressure of the PFC (the secondgas) will increase to 86 Torr. The air will diffuse out of the bubbleuntil it reaches osmotic equilibrium with the air dissolved in the blood(i.e., 760 Torr) and the partial pressure of the PFC will increase to100 Torr. The partial pressure of the PFC will act to counterbalance thepressure exerted due to the arterial pressure, halting shrinkage of thebubble, in each case, assuming that the permeability of the bubble tothe PFC is negligible.

When the surface tension or LaPlace pressure component of 100 Torr isadded (as discussed above with the air bubble), a total of 200 Torradditional pressure is exerted on the gas in the bubble. Again, thebubble will compress until and the pressure inside the bubble increasesto 960 Torr (partial pressure of air 864 and partial pressure of PFC96). The air will diffuse from the bubble until it reaches 760 Torr (inequilibrium with the concentration of air the dissolved in the blood)and the partial pressure of the PFC will increase to 200 Torr, where,again, the gas osmotic pressure induced by the PFC will act tocounterbalance the pressure exerted by the LaPlace pressure and thearterial pressure, again, assuming that the membrane permeability of thebubble to the PFC is negligible.

Similarly, if the partial pressure of air in the bubble is lower thanthe air tension in the surrounding liquid, the bubble will actually growuntil the PFC is sufficiently diluted by incoming air so that thepressure of air inside and the air tension outside of the bubble areidentical.

Thus, it can be seen has been shown that bubbles can be effectivelystabilized through the use of combinations of gases, since the correctcombination of gases will result in a gas osmotic pressure differentialthat can be harnessed to counterbalance the effects of the LaPlacepressure and the arterial pressure exerted on the a gas within thebubble in circulating blood.

Examples of particular uses of the microbubbles of the present inventioninclude perfusion imaging of the venous drainage system of the heart,the myocardial tissue, and determination of perfusion characteristics ofthe heart and its tissues during stress or exercise tests, or perfusiondefects or changes due to myocardial infarction. Similarly, myocardialtissue can be viewed after oral or venous administration of drugsdesigned to increase the blood flow to a tissue. Also, visualization ofchanges in myocardial tissue due to or during various interventions,such as coronary tissue vein grafting, coronary angioplasty, or use ofthrombolytic agents (TPA or streptokinase) can also be enhanced. Asthese contrast agents can be administered conveniently via a peripheralvein to enhance the visualization of the entire circulatory system, theywill also aid in the diagnosis of Deep Vein Thrombosis and in theability to ultrasonically monitor the fetus and the umbilical cord.

It should, however, be emphasized that these principles have applicationbeyond ultrasound imaging. Indeed, the present invention is sufficientlybroad to encompass the use of gas osmotic pressure to stabilize bubblesfor uses in any systems, including nonbiological applications.

In a preferred embodiment, the microbubbles of the present inventionhave a surfactant-based bubble membrane. However, the principles of theinvention can be applied to stabilize microbubbles of virtually anytype. Thus, mixed gases or vapors of the type described above canstabilize albumin based bubbles, polysaccharide based microbubbles,spray dried microsphere derived microbubbles, and the like. This resultis achieved through the entrapment, within the chosen microbubble, of acombination of gases, preferably a primary modifier gas or mixture ofgases that will dilute a gas osmotic agent to a partial pressure lessthan the gas osmotic agent's vapor pressure until the modifier gas willexchange with gases normally present in the external medium. The gasosmotic agent or agents are generally relatively hydrophobic andrelatively bubble membrane impermeable and also further possess theability to develop gas osmotic pressures greater than 50, 75, or 100Torr. In one preferred embodiment, the gas vapor pressure of the gasosmotic agent is preferably less than about 760 Torr at 37° C.,preferably less than about 750, 740, 730, 720, 710, or 700 Torr, and insome embodiments less than about 650, 600, 500, or 400 Torr.

In preferred embodiments, the vapor pressure of the primary modifier gasis at least 660 Torr at 37° C. and the vapor pressure of the gas osmoticagent is at least 100 Torr at 37° C. For in vivo imaging mean bubblediameters between 1 and 10 μm are preferred, with 3 to 5 μm mostpreferred. The invention may in one embodiment also be described as amixture of a first gas or gases and a second gas or gases withingenerally spherical membranes to form microbubbles, where the first gasand the second gas are respectively present in a molar ratio of about1:100, 1:75, 1:50, 1:30, 1:20, or 1:10 to about 1000:1, 500:1, 250:1,100:1, 75:1 or 50:1, and where the first gas has a vapor pressure of atleast about (760-x) mm Hg at 37° C., where x is the vapor pressure ofthe second gas at 37° C., and where the vapor pressure of each of thefirst and second gases is greater than about 75 or 100 mm Hg at 37° C.

Microbubbles prepared in accordance with one preferred embodiment of theinvention may also possess an additional advantageous property. In onesuch embodiment, mixtures of nonosmotic gases with osmotic stabilizinggases (or gas osmotic agents) are used to stabilize the resultant bubblesize distribution during and immediately after production. Upongeneration of the bubbles, the higher LaPlace pressure in smallerbubbles causes diffusion through the liquid phase to the lower LaPlacepressure larger bubbles. This causes the mean size distribution toincrease above the capillary dimension limit of 5 microns over time.This is called disproportionation. When a mixture of a nonosmotic gas(e.g., air) is used with an osmotic vapor (e.g., C₆F₁₄) a slightreduction in volume of the smaller bubbles, due to air leaving thebubble, concentrates the osmotic gas and increases its osmotic pressurethus retarding further shrinkage while the larger bubbles increase involume slightly, diluting the osmotic gas and retarding further growth.

An additional advantage of using a mixture of an extremely blood solublegases (e.g., 87.5% by volume CO₂) and an osmotic gas mixture (e.g., 28%C₆F₁₄ vapor+72% air) is that, when injected, these bubbles rapidlyshrink due to the loss of CO₂ to the blood. The bubbles, upon injection,will experience an 87.5% volume decrease due to loss of CO₂. This lossof CO₂ corresponds to a halving of the bubble diameter. Accordingly, onecan prepare larger diameter bubbles (e.g., 9 μm), using simplifiedmechanical means, that will shrink to below 5 microns upon injection. Ingeneral, such bubbles will initially be prepared where the first gas ispresent in a ratio of at least 1:1 with respect to the second gas,preferably at least 3:2, 2:1, 3:1, 4:1, 5:1, or 10:1. Where themicrobubble membrane is more permeable to the first gas than to thesecond gas (e.g., the membrane has respective permeabilities to thegases in a ratio of at least about 2:1, 3:1, 4:1, 5:1, or 10:1,preferably even higher, e.g., 20:1, 40:1, or 100:1), the bubblesadvantageously shrink from their original first diameter to an averagesecond diameter of 75% or less of their original diameter quite rapidly(e.g., within one, two, four, or five minutes). Then, when at least onerelatively membrane-permeable gas is present in the aqueous mediumsurrounding the microbubble, the bubble is preferably stabilized at orabout the second diameter for at least about 1 minute, preferably for 2,3, 4, or 5 minutes. In one preferred embodiment, the bubbles maintain asize between about 5 or 6 μm and 1 μm for at least 1, 2, 3, 4, or 5minutes, stabilized by a gas osmotic pressure differential. The gastension in the external liquid is preferably at least about 700 mm Hg.Moreover, a relatively membrane impermeable gas is also in themicrobubble to create such an osmotic pressure differential.

I. Microbubble Construction

A. The Aqueous or Other Liquid Phase

The external, continuous liquid phase in which the bubble residestypically includes a surfactant or foaming agent. Surfactants suitablefor use in the present invention include any compound or compositionthat aids in the formation and maintenance of the bubble membrane byforming a layer at the interface between the phases. The foaming agentor surfactant may comprise a single compound or any combination ofcompounds, such as in the case of co-surfactants.

Examples of suitable surfactants or foaming agents include: blockcopolymers of polyoxypropylene polyoxyethylene, sugar esters, fattyalcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters,hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol,nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxyethyl starch fatty acid esters, dextrans, dextran fatty acid esters,sorbitol, sorbitol fatty acid esters, gelatin, serum albumins, andcombinations thereof.

In the present invention, preferred surfactants or foaming agents areselected from the group consisting of phospholipids, nonionicsurfactants, neutral or anionic surfactants, fluorinated surfactants,which can be neutral or anionic, and combinations of such emulsifying orfoaming agents.

The nonionic surfactants suitable for use in the present inventioninclude polyoxyethylene-polyoxypropylene copolymers. An example of suchclass of compounds is Pluronic, such as Pluronic F-68. Also contemplatedare polyoxyethylene fatty acids esters, such as polyoxyethylenestearates, polyoxyethylene fatty alcohol ethers, polyoxyethylatedsorbitan fatty acid esters, glycerol polyethylene glycol oxystearate,glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols,ethoxylated castor oils, and the hydrogenated derivatives thereof, andcholesterol. Anionic surfactants, particularly fatty acids (or theirsalts) having 12 to 24 carbon atoms, may also be used. One example of asuitable anionic surfactant is oleic acid, or its salt, sodium oleate.

It will be appreciated that a wide range of surfactants can be used.Indeed, virtually any surfactant or foaming agent (including those stillto be developed) capable of facilitating formation of the microbubblescan be used in the present invention. The optimum surfactant or foamingagent or combination thereof for a given application can be determinedthrough empirical studies that do not require undue experimentation.Consequently, one practicing the art of the present invention shouldchoose the surfactant or foaming agents or combination thereof basedupon such properties as biocompatibility or their non-Newtonianbehavior.

The blood persistence of a contrast agent is inversely proportional tothe LaPlace pressure which is proportional to the surface tension of thebubble. Reduced surface tension, therefore, increases blood persistence.Surfactants that form ordered structures (multilaminar sheets and rods)in solution and produce non-Newtonian viscoelastic surface tensions areespecially useful. Such surfactants include many of the sugar basedsurfactants and protein or glycoprotein surfactants (including bovine,human, or other lung surfactants). One preferred type of such surfactanthas a sugar or other carbohydrate head group, and a hydrocarbon orfluorocarbon tail group. A large number of sugars are known that canfunction as head groups, including glucose, sucrose, mannose, lactose,fructose, dextrose, aldose, and the like. The tail group preferably hasfrom about 2 or 4 to 20 or 24 carbon atoms, and may be, for example, afatty acid group (branched or unbranched, saturated or unsaturated)covalently bound to the sugar through an ester bond. The surface tensionof bubbles produced with these surfactants greatly decreases as thesurface is compressed by shrinkage of the bubble (e.g., when the bubbleshrinks), and it is increased as the surface area of the bubble isincreased (e.g., when the bubble grows). This effect retardsdisproportionation, which leads to narrower size distribution and longerpersisting bubbles in the vial and in vivo. A preferred surfactantmixture that has the properties associated with non-Newtonianviscoelasticity includes a nonionic surfactant or other foamingsurfactant in combination with one of the non-Newtonian viscoelasticsurfactant such as one of the sugar esters (e.g. 2% Pluronic F-68 plus1% sucrose stearate). Often the ratio of the nonionic surfactant to thenon-Newtonian surfactant is from about 5:1 to about 1:5, with thesurfactants together (whether non-Newtonian or more conventional)comprising 0.5 to 8%, more preferably about 1 to 5% (w/v) of themicrobubble-forming liquid mixture.

The lowering of surface tension in small bubbles, counter to typicalLaPlace pressure, allows the use of more efficient gas osmotic agentssuch as higher molecular weight perfluorocarbons as the gas osmoticagent. With conventional surfactants, the higher molecular weight PFCswill condense at the high bubble pressures. Without these efficientsurfactants higher boiling less membrane permeable PFCs, e.g. C₆ F₁₄,would be extremely difficult.

One may also incorporate other agents within the aqueous phase. Suchagents may advantageously include conventional viscosity modifiers,buffers such as phosphate buffers or other conventional biocompatiblebuffers or pH adjusting agents such as acids or bases, osmotic agents(to provide isotonicity, hyperosmolarity, or hyposmolarity). Preferredsolutions have a pH of about 7 and are isotonic. However, when CO₂ isused as a first gas in a bubble designed to shrink rapidly to a firstsize, a basic pH can facilitate rapid shrinkage by removing CO₂ as itleaves the bubble, preventing a buildup of dissolved CO₂ in the aqueousphase.

B. The Gas Phase

A major aspect of the present invention is in the selection of the gasphase. As was discussed above, the invention relies on the use ofcombinations of gases to harness or cause differentials in partialpressures and to generate gas osmotic pressures, which stabilize thebubbles. The primary modifier gas is preferably air or a gas present inair. Air and/or fractions thereof are also present in normal blood andserum. Where the microbubbles are to be used in an environment differentfrom blood, the primary modifier gas is preferably selected from gasesnormally present in the external medium. Another criteria is the easewith which the primary modifier gas is diffused into or out of thebubbles. Typically, air and/or fractions thereof are also readilypermeable through conventional flexible or rigid bubble surfaces. Thesecriteria, in combination, allow for the rapid diffusion of the primarymodifier gas into or out of the bubbles, as required.

Modifier gases not present in the external medium can also be used.However, in this case the bubble will initially grow or shrink(depending on the relative permeability and concentrations of theexternal gases to the modifier) as the external gases replace theoriginal modifier gas. If, during this process, the gas osmotic agenthas not condensed, the bubble will remain stable.

The gas osmotic agent is preferably a gas that is less permeable throughthe bubble's surface than the modifier. It is also preferable that thegas osmotic agent is less soluble in blood and serum. Therefore, it willnow be understood that the gas osmotic agent can be a gas at room orbody temperature or it can ordinarily be a liquid at body temperature,so long as it has a sufficient partial or vapor pressure at thetemperature of use to provide the desired osmotic effect.

Accordingly, fluorocarbons or other compounds that are not gases at roomor body temperature can be used, provided that they have sufficientvapor pressure, preferably at least about 50 or 100 Torr at bodytemperature, or more preferably at least about 150 or 200 Torr. Itshould be noted that where the gas osmotic agent is a mixture of gases,the relevant measure of vapor pressure is the vapor pressure of themixture, not necessarily the vapor pressure of the individual componentsof the mixed gas osmotic agent.

It is also important that where a perfluorocarbon is used as the osmoticagent within a bubble, the particular perfluorocarbon does not condenseat the partial pressure present in the bubble and at body temperature.Depending on the relative concentrations of the primary modifier gas andthe gas osmotic agent, the primary modifier gas may rapidly leave thebubble causing it to shrink and concentrate the secondary gas osmoticagent. Such shrinking may occur until the gas osmotic pressure equalsthe external pressure on the bubble (maximum absolute arterial pressure)plus the LaPlace pressure of the bubble minus the air tension, or airsaturation tension, of the blood (essentially one atmosphere). Thus thecondensing partial pressure of the resulting gas mixture at 37° C. mustbe above the equilibrium partial pressure, discussed above, of theosmotic agent.

Representative fluorocarbons meeting these criteria and in increasingability to stabilize microbubbles are as follows:CCl₂F₂<CF₄, CHClF₂<C₄F₁₀, N(C₂F₅)₃<C₅F₁₂<C.₆F₁₄

Accordingly, it will be understood that PFC's with eight carbons atomsor fewer (37° C. vapor pressures greater than 80 mm Hg) are preferred.As will also be understood, however, it is possible to construct largermolecules with increased volatility through the addition of heteroatomsand the like. Therefore, the determination of the optimal secondary gasosmotic agent or gases agents is not size limited, but, rather, is basedupon its ability to retain its vapor phase at body temperature and whileproviding a gas osmotic pressure equal to at least the sum of thearterial and LaPlace pressures.

A listing of some compounds possessing suitable solubility and vaporpressure criteria is provided in Table I:

TABLE I perfluoro propanes, C₃F₈ perfluoro butanes, C₄F_(.10) perfluorocyclo butanes, C₄F_(.8) perfluoro pentanes, C₅F₁₂ perfluoro cyclopentanes, C_(.5)F₁₀ perfluoro methylcyclobutanes, C₅F₁₀ perfluorohexanes, C₆F_(.4) perfluoro cyclohexanes, C₆F₁₂ perfluoro methylcyclopentanes, C₆F₁₂ perfluoro dimethyl cyclobutanes, C₆F₁₂ perfluoroheptanes, C₇F_(.16) perfluoro cycloheptanes, C₇F₁₄ perfluoro methylcyclohexanes, C₇F₁₄ perfluoro dimethyl cyclopentanes, C₇F₁₄ perfluorotrimethyl cyclobutanes, C₇F₁₄ perfluoro triethylamines, N(C₂F₅)₃

It will be appreciated that one of ordinary skill in the art can readilydetermine other compounds that would perform suitably in the presentinvention that do not meet both the solubility and vapor pressurecriteria, described above. Rather, it will be understood that certaincompounds can be considered outside the preferred range in eithersolubility or vapor pressure, if such compounds compensate for theaberration in the other category and provide a superior insolubility orlow vapor pressure.

It should also be noted that for medical uses the gases, both themodifier gas and the gas osmotic agent, should be biocompatible or notbe physiologically deleterious. Ultimately, the microbubbles containingthe gas phase will decay and the gas phase will be released into theblood either as a dissolved gas or as submicron droplets of thecondensed liquid. It will be understood that gases will primarily beremoved from the body through lung respiration or through a combinationof respiration and other metabolic pathways in the reticuloendothelialsystem.

Appropriate gas combinations of the primary modifier and secondary gasescan be ascertained empirically without undue experimentation. Suchempirical determinations are described in the Examples.

When an efficient surfactant, e.g., bovine lung surfactant, is employedto produce a large diameter bubble with a low surface tension, theLaPlace pressure is very low. When perfluorooctylbromide (PFOB)saturated air is inside the bubble and the bubble is exposed to air or aliquid nearly saturated with air (e.g., equilibrated with air) the gasosmotic pressure is greater than the LaPlace pressure and therefore thebubble grows. With smaller diameter bubbles the LaPlace pressure ishigher and therefore the bubble shrinks and collapses. This shrinkage isat a reduced rate being driven by the difference between the LaPlacepressure minus reduced by the gas osmotic pressure. When small diameterbubbles are created by sonicating gas or gas vapor mixtures in a lowsurface tension surfactant solution, e.g., 2% pluronic F-68 plus 1%sucrose stearate, the time the bubbles persist in vitro, as observed bymicroscope, and in vivo as observed by Doppler ultrasound imaging of arabbit's kidney post intravenous injection, correlated with the abovegas osmotic pressure comparison.

In the rabbit kidney Doppler experiment (Example III), contrastenhancement was observed for up to 10 minutes with perfluorohexane/airmixtures in the bubbles compared with the instantaneous disappearance ofcontrast with pure air microbubbles. Thus, these perfluorochemicals arecapable of exerting gas osmotic pressures that nearly counterbalance theLaPlace pressure and create functional ultrasound microbubble contrastagents.

A surprising discovery was that mixtures of PFCs, e.g., C₄F₁₀ (as acombination modifier gas and a gas osmotic agent) saturated with C₆F₁₄vapor (as the main gas osmotic agent), can stabilize the bubble forlonger times than either component alone. This is because C₄ F₁₀ is agas at body temperature (and, thus, can act as both a modifier gas and agas osmotic agent) has a somewhat reduced membrane permeability and itis only slightly soluble in C₆F₁₄ at body temperature. In this situationthe gas osmotic pressures of both agents are added together, leading toincreased bubble persistence over that of air/C₆F₁₄ only mixtures. It ispossible that the condensing point of the longer persisting highermolecular weight C₆F₁₄ component is increased, allowing a larger maximumgas osmotic pressure to be exerted. Other mixtures of PFCs will performsimilarly. Preferred mixtures of PFCs will have ratios of 1:10 to 10:1,and include such mixtures as perfluorobutane/perfluorohexane andperfluorobutane/perfluoropentane. These preferred fluorochemicals can bebranched or straight chain.

As was discussed above, we have also discovered that mixtures ofnonosmotic gases in combination with the gas osmotic agent act tostabilize the size distribution of the bubbles before and afterinjection. Upon generation of the bubbles, the higher LaPlace pressuresin smaller bubbles causes diffusion through the liquid phase to thelower LaPlace pressure larger bubbles. This causes the mean sizedistribution to increase above the capillary dimension limit of 5microns with time. This is called disproportionation.

However, when a mixture of a modifier gases (e.g., air or carbondioxide) are used with a gas osmotic agent (e.g., C₆F₁₄) a slightreduction in volume of the smaller bubbles, due to one of the modifiergases leaving the bubble, will concentrate the osmotic gas and increasesits osmotic pressure, thus, retarding further shrinkage. On the otherhand, the larger bubbles will increase in volume slightly, diluting theosmotic gas and also retarding further growth.

An additional advantage of using a mixture of an extremely blood solublegas (e.g., 75% through 87.5% by volume CO₂) and an osmotic gas mixture(e.g. 28% C₆F₁₄ vapor and 72% air) is that when injected, these bubblesrapidly shrink due to the loss of CO₂ to the blood. Carbon dioxideleaves particularly fast due to a specific plasma enzyme that catalyzesits dissolution. An 87.5% volume decrease due to loss of CO₂ correspondswith a halving of the bubble diameter. Accordingly, larger can beproduced which will shrink to an appropriate size (i.e., 5 microns) uponinjection or exposure to a solution with a basic or alkaline pH.

Accordingly, we have discovered that through use of a gas that isrelatively hydrophobic and that has a relatively low membranepermeability, the rate of contrast particle decay can be reduced. Thus,through reducing the particle decay rate, the microbubbles' half livesare increased and contrast enhancement potential is extended.

II. Other Components.

It will be understood that other components can be included in themicrobubble formulations of the present invention. For example, osmoticagents, stabilizers, chelators, buffers, viscosity modulators, airsolubility modifiers, salts, and sugars can be added to fine tune themicrobubble suspensions for maximum life and contrast enhancementeffectiveness. Such considerations as sterility, isotonicity, andbiocompatibility may govern the use of such conventional additives toinjectable compositions. The use of such agents will be understood tothose of ordinary skill in the art and the specific quantities, ratios,and types of agents can be determined empirically without undueexperimentation.

III. Formation of the Microbubbles of the Present Invention.

There are a variety of methods to prepare microbubbles in accordancewith the present invention. Sonication is preferred for the formation ofmicrobubbles, i.e., through an ultrasound transmitting septum or bypenetrating a septum with an ultrasound probe including anultrasonically vibrating hypodermic needle. However, it will beappreciated that a variety of other techniques exist for bubbleformation. For example, gas injection techniques can be used, such asventuri gas injection.

Other methods for forming microbubbles include formation of particulatemicrospheres through the ultrasonication of albumin or other protein asdescribed in European Patent Application 0,359,246 by MolecularBiosystems, Inc.; the use of tensides and viscosity increasing agents asdescribed in U.S. Pat. No. 4,446,442; lipid coated, non-liposomal,microbubbles as is described in U.S. Pat. No. 4,684,479; liposomeshaving entrapped gases as is described in U.S. Pat. Nos. 5,088,499 and5,123,414; and the use of denatured albumin particulate microspheres asis described in U.S. Pat. No. 4,718,433. The disclosure of each of theforegoing patents and applications is hereby incorporated by reference.

Any of the above methods can be employed with similar success to entrainthe modifier gases and gas osmotic agents of the present invention.Moreover, it is expected that similar enhancement in longevity of thebubbles created will be observed through use of the invention.

Sonication can be accomplished in a number of ways. For example, a vialcontaining a surfactant solution and gas in the headspace of the vialcan be sonicated through a thin membrane. Preferably, the membrane isless than about 0.5 or 0.4 mm thick, and more preferably less than about0.3 or even 0.2 mm thick, i.e., thinner than the wavelength ofultrasound in the material, in order to provide acceptable transmissionand minimize membrane heating. The membrane can be made of materialssuch as rubber, Teflon, mylar, urethane, aluminized film, or any othersonically transparent synthetic or natural polymer film or film formingmaterial. The sonication can be done by contacting or even depressingthe membrane with an ultrasonic probe or with a focused ultrasound“beam.” The ultrasonic probe can be disposable. In either event, theprobe can be placed against or inserted through the membrane and intothe liquid. Once the sonication is accomplished, the microbubblesolution can be withdrawn from and vial and delivered to the patient.

Sonication can also be done within a syringe with a low powerultrasonically vibrated aspirating assembly on the syringe, similar toan inkjet printer. Also, a syringe or vial may be placed in andsonicated within a low power ultrasonic bath that focuses its energy ata point within the container.

Mechanical formation of microbubbles is also contemplated. For example,bubbles can be formed with a mechanical high shear valve (or doublesyringe needle) and two syringes, or an aspirator assembly on a syringe.Even simple shaking may be used. The shrinking bubble techniquesdescribed herein are particularly suitable for mechanically formedbubbles, having lower energy input than sonicated bubbles. Such bubbleswill typically have a diameter much larger than the ultimately desiredbiocompatible imaging agent, but can be made to shrink to an appropriatesize in accordance with the present invention.

In another method, microbubbles can be formed through the use of aliquid osmotic agent emulsion supersaturated with a modifier gas atelevated pressure introduced into in a surfactant solution. Thisproduction method works similarly to the opening of soda pop, where thegas foams upon release of pressure forming the bubbles.

In another method, bubbles can be formed similar to the foaming ofshaving cream, where perfluorobutane, freon, or another like materialthat boils when pressure is released. However, in this method it isimperative that the emulsified liquid boils sufficiently low or that itcontain numerous bubble nucleation sites so as to prevent superheatingand supersaturation of the aqueous phase. This supersaturation will leadto the generation of a small number of large bubbles on a limited numberof nucleation sites rather than the desired large number of smallbubbles (one for each droplet).

In still another method, dry void-containing particles or otherstructures (such as hollow spheres or honeycombs) that rapidly dissolveor hydrate, preferably in an aqueous solution, e.g., albumin, microfinesugar crystals, hollow spray dried sugar, salts, hollow surfactantspheres, dried porous polymer spheres, dried porous hyaluronic acid, orsubstituted hyaluronic acid spheres, or even commercially availabledried lactose microspheres can be stabilized with a gas osmotic agent.

For example, a spray dried surfactant solution can be formulated toobtain 5 micron or larger hollow spheres and packaged in a vial filledwith an osmotic gas or a desired gas mixture as described herein. Thegas will diffuse into the spheres. Diffusion can be aided by pressure orvacuum cycling. When reconstituted with a sterile solution the sphereswill rapidly dissolve and leave osmotic gas stabilized bubbles in thevial. In the alternative, a lyophilized cake of surfactant and bulkingreagents produced with a fine pore structure can be placed in a vialwith a sterile solution and a head spaced with an osmotic gas mixture.The solution can be frozen rapidly to produce a fine ice crystalstructure and, therefore, upon lyophilization produces fine pores (voidswhere the ice crystals were removed).

Alternatively, any dissolvable or soluble void-forming structures may beused. In this embodiment, where the void-forming material is not madefrom or does not contain surfactant, both surfactant and liquid aresupplied into the container with the structures and the desired gas orgases. Upon reconstitution these voids trap the osmotic gas and, withthe dissolution of the solid cake, form microbubbles with the gas orgases in them.

It will be appreciated that kits can be prepared for use in making themicrobubble preparations of the present invention. These kits caninclude a container enclosing the gas or gases described above forforming the microbubbles, the liquid, and the surfactant. Alternatively,the container can contain the void forming material and the gas orgases, and the surfactant and liquid can be added to form themicrobubbles. Alternatively, the surfactant can be present with theother materials in the container, and only the liquid needs to be addedin order to form the microbubbles. Where a material necessary forforming the microbubbles is not already present in the container, it canbe packaged with the other components of the kit, preferably in a formor container adapted to facilitate ready combination with the othercomponents of the kit.

The container used in the kit may be of the type described elsewhereherein. In one embodiment, the container is a conventional septum-sealedvial. In another, it has a means for directing or permitting applicationof sufficient bubble forming energy into the contents of the container.This means can comprise, for example, the thin web or sheet describedpreviously.

Any of the microbubble preparations of the present invention may beadministered to a vertebrate, such as a bird or a mammal, as a contrastagent for ultrasonically imaging portions of the vertebrate. Preferably,the vertebrate is a human, and the portion that is imaged is thevasculature of the vertebrate. In this embodiment, a small quantity ofmicrobubbles (e.g., 0.1 ml/Kg based on the body weight of thevertebrate) is introduced intravascularly into the animal. Otherquantities of microbubbles, such as from about 0.005 ml/Kg to about 1.0ml/Kg, can also be used. Imaging of the heart, arteries, veins, andorgans rich in blood, such as liver, lungs, and kidneys can beultrasonically imaged with this technique.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, exemplary ofpreferred methods of practicing the present invention and are notlimiting of the scope of the invention or the claims appended hereto.

EXAMPLE I Preparation of Microbubbles Through Sonication

Microbubbles with an average number weighted size of 5 microns wereprepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants, air as a modifiergas and perfluorohexane as the gas osmotic agent.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 mlvial. The vial had a remaining head space of 0.7 ml initially containingair. Air saturated with perfluorohexane vapor (220 torr ofperfluorohexane with 540 torr of air) at 25 degrees C. was used to flushthe headspace of the vial. The vial was sealed with a thin 0.22 mmpolytetrafluoroethylene (PFFE) septum. The vial was turned horizontally,and a ⅛″ (3 mm) sonication probe attached to a 50 watt sonicator modelVC₅₀, available from Sonics & Materials was pressed gently against theseptum. In this position, the septum separates the probe from thesolution. Power was then applied to the probe and the solution wassonicated for 15 seconds, forming a white solution of finely dividedmicrobubbles, having an average number weighted size of 5 microns asmeasured by Horiba LA-700 laser light scattering particle analyzer.

EXAMPLE II Measurement of In-Vitro Size of Microbubbles

The in-vitro size of the microbubbles prepared in Example I was measuredby laser light scattering. Studies of bubbles were conducted where themicrobubbles were diluted into a 4% dextrose water solution (1:50)circulating through a Horiba LA-700 laser light scattering analyzer. Theaverage microbubbles size was 5 microns and doubled in size in 25minutes.

Interestingly, microbubbles prepared through the same method in ExampleI without the use of a gas osmotic agent (substituting air for theperfluorohexane/air mixture) had an average size of 11 microns and gaveonly background readings on the particle analyzer at 10 seconds.

EXAMPLE III Measurement of In-Vivo Lifetime of Microbubbles

The lifetimes of microbubbles prepared in accordance with Example I weremeasured in rabbits through injecting 0.2 ml of freshly formedmicrobubbles into the marginal ear vein of a rabbit that was underobservation with a Accuson 128XP/5 ultrasound imaging instrument with a5 megahertz transducer. Several tests were conducted, during whichimages of the heart, inferior vena cava/aorta, and kidney were obtainedwhile measuring the time and extent of the observable contrastenhancement. The results are presented in the following Table II:

TABLE II TIME TO MINIMUM TIME MAX. USABLE TIME TO NO ORGAN DOSEINTENSITY INTENSITY ENHANCEMENT Heart 0.1 ml/Kg 7–10 sec. 8–10 min. 25min IVC/Aorta 0.1 ml/Kg 7–10 sec. 8–10 min. 25 min Kidney 0.1 ml/Kg 7–10sec. 8–10 min  25 minIn Table III, a comparison of microbubbles prepared in an identicalfashion without the use of an osmotic gas is presented (only air wasused). Note that sporadic reflections were observed only in the rightheart ventricle during the injection but disappeared immediately postdosing.

TABLE III TIME TO TIME TO MINIMUM MAXIMUM USABLE TIME TO NO ORGAN DOSEINTENSITY INTENSITY ENHANCEMENT Heart 0.1 ml/Kg 0 0 0 IVC/Aorta 0.1ml/Kg 0 0 0 Kidney 0.1 ml/Kg 0 0 0The use of an osmotic or gas osmotic agent dramatically increased thelength of time for which microbubbles are visible.

EXAMPLE IV Preparation of Mixed Osmotically Stabilized Microbubbles

Microbubbles with an average number weighted size of 5 microns wereprepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants and mixtures ofperfluorohexane and perfluorobutane as the gas osmotic agents.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl and 2% Pluronic F-68 was added to a 2.0 ml vial. The vial had aremaining head space of 0.7 ml, initially containing air. An osmotic gasmixture of perfluorohexane, 540 Torr and perfluorobutane at 220 Torr wasused to flush the headspace before sealing with a thin 0.22 mm PTFEseptum. The vial was sonicated as in Example I, forming a white solutionof finely divided microbubbles, having an average particle size of 5microns as measured by a Horiba LA-700 laser light scattering particleanalyzer. This procedure was repeated twice more, once with pureperfluorobutane and then with a 540 Torr air+220 Torr perfluorohexanemixture. Vascular persistence of all three preparations was determinedby ultrasound imaging of a rabbit post I.V. injection and are listedbelow:

1.5 minutes perfluorobutane   2 minutes perfluorohexane + air   3minutes perfluorbutane + perfluorohexane

The mixture of perfluorocarbons persisted longer than either agentalone.

EXAMPLE V Preparation of Gas Osmotically Stabilized Microbubbles fromSoluble Spray Dried Spheres

Gas osmotically stabilized microbubbles were prepared by dissolvinghollow spray dried lactose spheres, filled with an air perfluorohexanevapor mixture, in a surfactant solution.

Spray dried spheres of lactose with a mean diameter of approximately 100micron and containing numerous 10 to 50 micron cavities, was obtainedfrom DMV International under the trade name of Pharmatose DCL-11. Ninetymilligrams of the lactose spheres was placed in a 2.0 ml vial. Theporous spheres were filled with a mixture of 220 Torr perfluorohexaneand 540 Torr air by cycling the gas pressure in the vial between oneatmosphere and ½ atmosphere a total of 12 times over 5 minutes. Asurfactant solution containing 0.9% sodium chloride, 2% Pluronic-F₆₈ and1% sucrose stearate was warmed to approximately 45° C., to speed thedissolution of the lactose, before injecting 1.5 ml of the warmedsolution into the vial. The vial was then gently agitated by inversionfor approximately 30 seconds to dissolve the lactose before injectingthe microbubbles thus prepared into the Horiba LA-700 particle analyzer.A 7.7 micron volume weighted median diameter was measured approximatelyone minute after dissolution. The diameter of these microbubblesremained nearly constant, changing to a median diameter of 7.1 micronsin 10 minutes. When the experiment was repeated with air filled lactose,the particle analyzer gave only background readings one minute afterdissolution, thus demonstrating that gas osmotically stabilizedmicrobubbles can be produced by the dissolution of gas-filledcavity-containing structures.

EXAMPLE VI Preparation of Larger Bubbles that Shrink to a Desired Size

Microbubbles with an average volume weighted size of 20 micronsshrinking to 2 microns were prepared by sonication of an isotonicaqueous phase containing 2% Pluronic F-68 as the surfactant, CO₂ as adiluent gas and perfluorohexane as the gas osmotic agent.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 mlvial. The vial had a remaining head space of 0.7 ml initially containingair. A mixture of air saturated with perfluorohexane at 25 degrees C.diluted by a factor of 10 with CO₂ (684 Torr CO₂+54 Torr air+22 Torrperfluorohexane) was used to flush the head space. The vial was sealedwith a thin 0.22 mm PTFE septum. The vial was sonicated as in Example I,forming a white solution of finely divided microbubbles, having anaverage particle size of 28 microns as measured by Horiba LA-700 laserlight scattering analyzer. In the 4% dextrose+0.25 mM NaOH solution ofthe Horiba, the average bubble diameter rapidly shrank in 2 to 4 minutesfrom 28 microns to 5 to 7 microns, and then remained relativelyconstant, reaching 2.6 micron after 27 minutes. This is because the CO₂leaves the microbubbles by dissolving into the water phase.

EXAMPLE VII Perfluoroheptane Stabilized Microbubble In Vitro Experiment

Microbubbles were prepared as in Example I above employingperfluoroheptane saturated air (75 torr plus 685 torr air) and weremeasured as in Example II above. The average number weighted diameter ofthese microbubbles was 7.6 micron, one minute after circulation, and 2.2microns after 8 minutes of circulation. This persistence, compared tothe near immediate disappearance of microbubbles containing only air,demonstrates the gas osmotic stabilization of perfluoroheptane.

EXAMPLE VIII Perfluorotripropyl Amine Stabilized Microbubble In VivoExperiment

Microbubbles were prepared as in Example I above, employingperfluorotripropyl amine saturated air and were assessed as in ExampleIII above. The usable vascular persistence of these microbubbles wasfound to be 2.5 minutes, thus demonstrating the gas osmoticstabilization of perfluorotripropyl amine.

EXAMPLE IX Effect of a Non Newtonian Viscoelastic Surfactant—SucroseStearate

Microbubbles were prepared as in Example I above employing 0.9% NaCl, 2%Pluronic F-68 and 2% sucrose stearate as the surfactant and withperfluoropropane saturated air and perfluorohexane saturated air in theheadspace. These two preparations were repeated with the same surfactantsolution minus sucrose stearate. All four microbubble preparations wereassessed as in Example III above. The usable vascular persistence ofthese microbubbles are listed below:

2% Pluronic F-68+2% Sucrose Stearate Persistence

-   -   2 minutes perfluoropropane    -   4 minutes perfluorohexane

2% Pluronic F-68 Only Persistence

-   -   2 minutes perfluoropropane    -   1 minute perfluorohexane

As seen above, the reduced surface tension made possible by thenon-Newtonian viscoelastic properties of sucrose stearate prevented theless volatile perfluorohexane from condensing, allowing perfluorohexanemicrobubbles of longer persistence to be produced.

The foregoing description details certain preferred embodiments of thepresent invention and describes the best mode contemplated. It will beappreciated, however, that no matter how detailed the foregoing appearsin text, the invention can be practiced in many ways and the inventionshould be construed in accordance with the appended claims and anyequivalents thereof.

1. A microbubble for use in diagnostic imaging in a liquid mediumwherein the microbubble transports physiological gases in vivo whereinthe microbubble comprises a membrane and at least one fluorocarbon gasand at least one modifier gas wherein the microbubble grows and shrinksto maintain osmotic equilibrium with the physiological gas saturation ofthe surrounding liquid medium.
 2. The microbubble of claim 1 wherein themodifier gas saturation level changes in the bubble as the microbubblecirculates in the bloodstream of the patient.
 3. The microbubble ofclaim 1 wherein the surrounding liquid medium is blood.
 4. Themicrobubble of claim 1 wherein the modifier gas is at least one gasselected from the group consisting of oxygen, nitrogen and carbondioxide.
 5. The microbubble of claim 1 wherein the microbubble growswhen the modifier gas of the microbubble exchanges with gases present inthe surrounding liquid medium.
 6. The microbubble of claim 5 wherein thegas present in the surrounding liquid medium is oxygen.
 7. Themicrobubble of claim 5 wherein the gas present in the surrounding liquidmedium is air.
 8. The microbubble of claim 1 wherein the at least onefluorocarbon gas is selected from the group consisting ofperfluoropropanes, perfluorobutanes, perfluorocyclobutanes,perfluoropentanes, perfluorocyclopentanes, perfluoromethylcyclopentanes,perfluorohexanes, perfluorocyclohexanes, perfluoromethylcyclopentanes,perfluorodimethylcyclobutanes, perfluoroheptanes,perfluorocycloheptanes, perfluoromethylcyclohexanes,perfluorodimethylcyclopentanes, perfluorotrimethylcyclobutanes, andperfluorotriethylamines.
 9. A microbubble for use in diagnostic imagingin a liquid medium, wherein the microbubble transports physiologicalgases and wherein the microbubble comprises at least one fluorocarbongas and at least one modifier gas comprising oxygen wherein themicrobubble grows or-shrinks in the surrounding liquid medium.
 10. Themicrobubble composition of claim 9 wherein the fluorocarbon gas isperfluorohexane.
 11. The microbubble of claim 9 wherein the microbubblegrows in diameter to maintain osmotic equilibrium of oxygen within themicrobubble with the oxygen in the surrounding liquid medium.
 12. Themicrobubble of claim 9 wherein the surrounding liquid medium is blood.13. The microbubble of claim 9 wherein the first fluorocarbon gas isselected from the group consisting of perfluoropropanes,perfluorobutanes, perfluorocyclobutanes, perfluoropentanes,perfluorocyclopentanes, peffluoromethylcyclopentanes, perfluorohexanes,perfluorocyclohexanes, perfluoromethylcyclopentanes,perfluorodimethylcyclobutanes, perfluoroheptanes,perfluorocycloheptanes, perfluoromethylcyclohexanes,perfluorodimethylcyclopentanes, perfluorotrimethylcyclobutanes, andperfluorotriethylamines.
 14. The microbubble of claim 9 wherein themicrobubble further comprises a membrane.
 15. The microbubble of claim10 wherein the microbubble further comprises a membrane.
 16. Amicrobubble composition for use in diagnostic imaging in a liquid mediumwherein the microbubble comprises a membrane and transportsphysiological gases in vivo wherein the microbubble comprises at leastone fluorocarbon gas and at least one modifier gas wherein themicrobubble first shrinks as a result of loss of the modifier gas to thesurrounding liquid medium and then grows as the microbubble gainsosmotic equilibrium with the physiological gas saturation of thesurrounding liquid medium.
 17. The microbubble composition of claim 16wherein the modifier gas is selected from the group consisting ofoxygen, nitrogen and carbon dioxide.
 18. The microbubble composition ofclaim 16 wherein the transported physiological gas is oxygen.
 19. Themicrobubble composition of claim 16 wherein the fluorocarbon gas isselected from the group consisting of perfluoropropanes,perfluorobutanes, perfluorocyclobutanes, perfluoropentanes,perfluorocyclopentanes, perfluoromethylcyclopentanes, perfluorohexanes,perfluorocyclohexanes, perfluoromethylcyclopentanes,perfluorodimethylcyclobutanes, perfluoroheptanes,perfluorocycloheptanes, perfluoromethylcyclohexanes,perfluorodimethylcyclopentanes, perfluorotrimethylcyclobutanes, andverfluorotriethylamines.